1,4-Benzodiazepine peripheral cholecystokinin (CCK-A) receptor agonists.

A series of 1,4-benzodiazepines, N-1-substituted with an N-isopropyl-N-phenylacetamide moiety, was synthesized and screened for CCK-A agonist activity. In vitro agonist activity on isolated guinea pig gallbladder along with in vivo induction of satiety following intraperitoneal administration in a rat feeding assay was demonstrated.

Optimization of 3-(1H-indazol-3-ylmethyl)-1,5-benzodiazepines as potent, orally active CCK-A agonists.

We previously described a series of 3-(1H-indazol-3-ylmethyl)-1,5-benzodiazepine CCK-A agonists exemplified by compound 1 (GW 5823), which is the first reported binding selective CCK-A full agonist demonstrating oral efficacy in a rat feeding model. In this report we describe analogs of compound 1 designed to explore changes to the C3 and N1 pharmacophores and their effect on agonist activity and receptor selectivity. Agonist efficacy in this series was affected by stereoelectronic factors within the C3 moiety. Binding affinity for the CCK-A vs CCK-B receptor showed little dependence on the structure of the C3 moiety but was affected by the nature of the second substituent at C3. Structure-activity relationships at the N1-anilidoacetamide "trigger" moiety within the C3 indazole series were also investigated. Both agonist efficacy and binding affinity within this series were modulated by variation of substituents on the N1-anilidoacetamide moiety. Evaluation of several analogs in an vivo mouse gallbladder emptying assay revealed compound 1 to be the most potent and efficacious of all the analogs tested. The pharmacokinetic and pharmacodynamic profile of 1 in rats is also discussed.

Discovery of 1,5-benzodiazepines with peripheral cholecystokinin (CCK-A) receptor agonist activity (II): Optimization of the C3 amino substituent.

Analogs of the previously reported 1,5-benzodiazepine peripheral cholecystokinin (CCK-A) receptor agonist 1 were prepared which explore substitution and/or replacement of the C-3 phenyl urea moiety. Agonist efficacy on the isolated guinea pig gallbladder (GPGB) was retained with a variety of substituted ureas and amide analogs. Three compounds were identified which were orally active in the mouse gallbladder emptying assay (MGBE). The 2-indolamide (52) and N-(carboxymethyl)-2-indolamide (54) derivatives had improved affinity for the human CCK-A receptor but reduced agonist efficacy on the GPGB. Neither indolamide was orally active in a rat feeding assay. In contrast, the (3-carboxyphenyl)urea derivative (29, GW7854) had moderately increased affinity for the human CCK-B receptor but was a potent full agonist on the GPGB and was orally active in both the MGBE and rat feeding assays. GW7854 was a full agonist (EC50 = 60 nM) for calcium mobilization on CHO K1 cells expressing hCCK-A receptors and a potent antagonist of CCK-8 (pA2 = 9.1) on CHO K1 cells expressing hCCK-B receptors. GW7854 is a potent mixed CCK-A agonist/CCK-B antagonist which is orally active in two in vivo models of CCK-A-mediated agonist activity.

3-[2-(N-phenylacetamide)]-1,5-benzodiazepines: orally active, binding selective CCK-A agonists.

A series of modifications were made to the C-3 substituent of the 1,5-benzodiazepine CCK-A agonist 1. Replacement of the inner urea NH and addition of a methyl group to generate a C-3 quaternary carbon resulted in acetamide 6, which showed CCK-A receptor binding selectivity and sub-micromolar agonist activity in vitro. Benzodiazepine 6 was active in an in vivo mouse gallbladder emptying assay and represents a novel orally active, binding selective CCK-A agonist.

Structure activity relationships of a series of buspirone analogs at alpha-1 adrenoceptors: further evidence that rat aorta alpha-1 adrenoceptors are of the alpha-1D-subtype.

The activity of a series of busprione analogs at recombinant and rat thoracic aorta alpha-1 adrenoceptors was investigated. Compound affinity for recombinant alpha-1A, alpha-1B and alpha-1D adrenoceptors from human and animal sources was determined by radioligand binding assays using membranes prepared from rat-1 fibroblasts expressing recombinant receptors with ( +/- )-[125l]iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone as the radioligand. Compound affinity and functional activity at rat aortic alpha-1 adrenoceptors were determined using endothelium denuded rings contracted with phenylephrine. BMY 7378 ¿8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]-ehtyl)-8-azaspiro [4,5]decane-7,9-dione dihydrochloride¿ and MDL 73005EF ¿8-[2-(1,4-benzodioxan-2-ylmethylamino) ethyl]-8-azaspiro[4,5]decane-7,9-dione hydrochloride¿ were found to have significant selectivity for the alpha-1D-subtype and were high affinity antagonists of the alpha-1 adrenoceptors in the rat aorta. Leverage plot analysis of affinities of the buspirone analogs and a series of structurally diverse alpha-1 antagonists for recombinant alpha-1 adrenoceptors and rat aorta alpha-1 adrenoceptors demonstrate that the alpha-1 adrenoceptors in the rat aorta are predominantly of the alpha-1D subtype.

Discovery of 1,5-benzodiazepines with peripheral cholecystokinin (CCK-A) receptor agonist activity. 1. Optimization of the agonist "trigger".

Directed screening of compounds selected from the Glaxo registry file for contractile activity on the isolated guinea pig gallbladder (GPGB) identified a series of 1,5-benzodiazepines with peripheral cholecystokinin (CCK) receptor agonist activity. Agonist efficacy within this series was modulated by variation of substituents on the N1-anilinoacetamide moiety. Remarkably, a single methyl group confers agonist activity, with an N-isopropyl substituent providing optimal efficacy. Hydrophilic substituents on the anilino nitrogen abolish agonist activity or produce antagonists of CCK. In contrast, hydrophilic electron-donating groups at the para-position of the anilino ring enhance or maintain in vitro and in vivo agonist activity. Despite decreased affinity for the human CCK-A receptor, relative to CCK-8, some of these compounds are equipotent to CCK as anorectic agents in rats following intraperitoneal administration.

CCK-A receptor selective antagonists derived from the CCK-A receptor selective tetrapeptide agonist Boc-Trp-Lys(Tac)-Asp-MePhe-NH2 (A-71623).

Analogs of the CCK-A receptor selective agonist Boc-Trp-Lys(Tac)-Asp-MePhe-NH2 (A-71623) were prepared in which the lysine residue was replaced with L-4-aminophenylalanine and D-or L-3-aminophenylalanine. These new analogs were moderately potent antagonists of CCK-8 in the isolated guinea pig gallbladder with exceptional CCK-A receptor selectivity as evaluated in membrane preparations from CHO K1 cells stably transfected with human CCK-A and CCK-B receptors.

Trophic control of lung development by sympathetic neurons: effects of neonatal sympathectomy with 6-hydroxydopamine.

The onset of peripheral sympathetic neuronal function is thought to provide trophic regulatory signals for development of adrenergic target tissues. In the current study, we examined the effects on lung development of neonatal sympathectomy with 6-hydroxydopamine. The completeness of the lesion and effectiveness in reducing sympathetic input to the tissue were confirmed by direct measurement of norepinephrine levels and turnover. Despite the denervation, no evidence of beta-receptor up-regulation was found; in fact, receptor binding sites tended to be reduced throughout development. The cyclic AMP response to isoproterenol challenge was initially suppressed in the lesioned animals, but became supersensitive even in the face of reduced receptor binding capabilities. Evidence was also obtained for ontogenetic abnormalities in the ornithine decarboxylase/polyamine system, which is partially controlled by beta-adrenergic input and which regulates macromolecule synthesis in replicating and differentiating cells. Eventually, the alterations were reflected in aberrant developmental patterns of DNA, RNA and protein in the lung. These results indicate that sympathetic neurons influence the biochemical development of the lung and may serve to program permanently the relationships among receptor sites, receptor coupling to cellular function, and control of cell maturation.

Role of sympathetic neurons in biochemical and functional development of the kidney: neonatal sympathectomy with 6-hydroxydopamine.

Renal sympathetic function develops over the first 3 weeks of postnatal life in the rat. In the current study, the effects of neonatal sympathectomy with 6-hydroxydopamine were examined on renal biochemical and functional development. The completeness and persistence of sympathetic nerve loss were confirmed by direct measurement of norepinephrine levels and turnover. Evidence was obtained for adverse effects on cellular maturation, as shown by perturbations in the ornithine decarboxylase/polyamine system, which is controlled partially by beta adrenergic input and which regulates macromolecule synthesis in developing cells. A later phase of 6-hydroxydopamine-induced alterations in renal development was seen during the period in which synaptogenesis is prominent and sympathetic tone is high (end of the 2nd postnatal week to end of the 3rd week): the denervated kidneys displayed supersensitivity of beta adrenergically mediated cyclic AMP responses without changes in receptor binding. The alterations in biochemical indices of cellular maturation were accompanied by abnormalities of renal function. 6-Hydroxydopamine caused an increase in the fractional excretion of sodium and deficits in physiological responsiveness of the kidney to a vasopressin analog. Later on, alterations in glomerular filtration rate and basal urinary osmolality also were prominent. These results indicate that neonatal sympathectomy has an adverse effect on the biochemical and functional development of the kidney.

Do sympathetic neurons coordinate cellular development in the heart and kidney? Effects of neonatal central and peripheral catecholaminergic lesions on cardiac and renal nucleic acids and proteins.

Sympathetic nerve activity has been hypothesized to set the timing of cellular maturational events in target tissues. In the current study, this hypothesis was tested by lesioning catecholamine pathways in the periphery and central nervous system through the use of subcutaneous or intracisternal injections of 6-hydroxydopamine. Systemically administered 6-hydroxydopamine completely depleted peripheral norepinephrine. The central treatment completely ablated the developmental rise in brain norepinephrine and dopamine and had little effect on peripheral norepinephrine levels, but has been shown to reduce sympathetic tone. In both the heart and the kidney, either type of lesion resulted in deficits in cell acquisition (DNA) with some evidence of compensatory increases in other macromolecules involved in cell enlargement (particularly RNA), thus maintaining the tissue growth rate at only slightly subnormal levels. The peak effect was always seen during the stages at which sympathetic neuronal synaptogenesis and impulse activity ordinarily undergo their most rapid development. Most of the 6-hydroxydopamine-induced differences in nucleic acids lessened or disappeared toward weaning, and thus these data support the view that sympathetic neuronal input influences the timing of maturational control of macromolecules, but not their final set-point. In combination with earlier studies showing termination of DNA synthesis caused by exposure of heart and kidney acutely to high levels of catecholamines, the results suggest that neuronal activity provides a biphasic signal, with positive trophic effects predominating during early development when sympathetic tone is low, and negative effects appearing when sympathetic tone is elevated during the late preweanling stage.

Biochemical determinants of growth sparing during neonatal nutritional deprivation or enhancement: ornithine decarboxylase, polyamines, and macromolecules in brain regions and heart.

In order to elucidate the biochemical mechanisms operating to protect the brain from growth retardation in response to nutritional deprivation, comparisons were made of markers of cellular development in brain regions (cerebellum, cerebral cortex, midbrain + brainstem) and in a tissue which is not spared (heart). Nutritional status of neonatal rats was manipulated by increasing or decreasing the litter size beginning at birth, and development of DNA, RNA, and proteins followed throughout the neonatal period. In addition, we assessed the activity and levels of ornithine decarboxylase and its metabolic products, the polyamines, which are known to coordinate macromolecule synthesis in immature tissue and to provide an early index of perturbed development. Cardiac ornithine decarboxylase and polyamines were altered within 48 h of initiating the changes in litter size, and the direction and magnitude of these biochemical effects were predictive of subsequent impairment or enhancement of organ growth and of cellular development. All three brain regions were buffered from growth alterations relative to the heart, but the cerebellum, which undergoes major phases of cell replication later than the other two regions, was somewhat less protected. The spared brain regions also showed evidence of compensatory hypertrophy in nutritional deprivation (increased protein/DNA ratio) which accounts for maintenance of growth in the presence of reduced cell numbers. Thus, brain growth sparing involves specific cellular responses which are dependent on the maturational profile of each brain region.

Beta adrenergic control of macromolecule synthesis in neonatal rat heart, kidney and lung: relationship to sympathetic neuronal development.

The sympathetic nervous system has been hypothesized to coordinate the timing of cellular development in peripheral tissues. In the current study, we evaluated the relationships among the ontogeny of sympathetic projections to peripheral organs, the patterns of macromolecule synthesis in those organs and the reactivity of synthetic processes to beta adrenergic stimulation by isoproterenol. The major developmental rise in norepinephrine concentration and turnover, as well as in numbers of beta receptors, occurred during the second to fourth postnatal weeks in renal and lung sympathetic pathways and slightly earlier in the cardiac-sympathetic axis. The developmental decline in DNA synthesis in heart, kidney and lung coincided with the maturation of sympathetic projections. Direct stimulation of beta receptors by the in vivo administration of isoproterenol caused acute reductions in DNA synthesis in an age-dependent manner. In the heart, isoproterenol was first able to suppress DNA synthesis at 5 days of age and a maximal effect was seen at 9 days; this early phase was characterized by a rapid time constant of coupling of beta receptors to the DNA effect (maximal effect at 6 h after isoproterenol). Reactivity was lessened by 12 days of age and thereafter displayed a longer time constant (maximal effect at 12-24 h). Reactivity of DNA synthesis to isoproterenol challenge was slightly different in kidney and lung (detectable by 2 days of age), but bore similar developmental characteristics to the pattern in the heart (peak of reactivity at 9 days and a decline in reactivity and lengthening of the time constant after 16 days).(ABSTRACT TRUNCATED AT 250 WORDS)

Development of [3H]nicotine binding sites in brain regions of rats exposed to nicotine prenatally via maternal injections or infusions.

The fetal and postnatal development of binding sites for [3H]nicotine was examined in brain regions of normal rats and rats whose mothers received nicotine injections or infusions, starting before fetal implantation (gestational day 4) and continuing to gestational day 20. The normal ontogenetic pattern of binding indicated a small but detectable concentration of sites during late gestation, and a substantial increase after birth, primarily during the period in which the majority of cholinergic synapses is forming. The adult pattern of regional selectivity of binding capabilities, namely midbrain + brainstem greater than cerebral cortex much greater than cerebellum, was not present at birth, but rather developed over the ensuing 3 weeks postpartum. Fetal exposure to nicotine produced an elevation in binding detectable during the course of drug exposure (gestational day 18), a finding similar to nicotine's effects in mature brain. However, examination of the subsequent developmental pattern of [3H]nicotine binding indicated a generalized disruption of receptor acquisition, in that alterations persisted far beyond the period in which drug exposure was terminated. The greatest effect was seen in a region relatively poor in receptor sites (cerebellum), and a larger stimulation was obtained with injected vs. infused nicotine. Because the cerebellum is the primary target for disruption of cellular development by prenatal nicotine exposure, these results are consistent with a primary teratologic action of the drug rather than direct effects on development of [3H]nicotine receptors.

Effects of prenatal nicotine exposure on biochemical development of rat brain regions: maternal drug infusions via osmotic minipumps.

The effects of a continuous 16-day gestational exposure to nicotine on brain development were examined in the offspring of dams who received a minipump implant on the 4th day of gestation. Maternal viability was unaffected and weight gain was only reduced slightly, but nearly half the dams failed to give birth; dams delivering pups had normal litter sizes. Examination of fetal macromolecules on the 18th day of gestation revealed specific deficits in cell number (DNA) in developing brain tissue as opposed to the rest of the fetus, accompanied by parallel shortfalls in other macromolecules (RNA, protein). After birth, brain development in the nicotine-exposed animals showed persistent abnormalities in the timing of maturational events, with elevated levels of ornithine decarboxylase (an enzymatic marker related to cellular maturation) detectable in all brain regions. Subsequent effects on macromolecules were highly selective regionally, with clear distinctions between areas in which neuronal replication occurs relatively late (cerebellum) compared to early-developing regions (midbrain plus brainstem). Differences apparent between the effects of infused maternal nicotine and those noted previously in studies with nicotine injections indicate that the drug does exert primary effects on developing neural tissues, but that other factors associated with the injection route (such as hypoxia and ischemia consequent to acute effects of nicotine) can interact with the drug to influence brain cell maturation.

Do catecholamines contribute to the effects of neonatal hypoxia on development of brain and heart? Influence of concurrent alpha-adrenergic blockade on ornithine decarboxylase activity.

Hypoxia in the neonate releases catecholamines from the adrenal medulla, a response which is necessary to survive. This study examines whether a similar dependence exists for the ability of brain and heart tissue to recover from hypoxia-induced damage, as assessed by measurements of ornithine decarboxylase (ODC) activity. Hypoxia at either 1 day or 8 days of age produced a subsequent elevation of brain ODC which persisted for 1 week, a pattern known to be associated with recovery from tissue damage and delayed cellular maturation. Pretreatment of the rats with phenoxybenzamine, an alpha-receptor blocking agent, resulted in attenuation of the long-term ODC response, but did not interfere with effects on the enzyme during the hypoxia itself. In the heart, hypoxia at 8 days of age displayed similar effects, with long-term ODC elevations which were attenuated by phenoxybenzamine. Hypoxia at 1 day of age also produced long-term heart ODC stimulation, but in this case the effect was exacerbated by phenoxybenzamine, an effect consistent with the greater dependence of cardiac tissue on alpha-receptor-mediated responses to hypoxia at that age. These results suggest that alpha-receptor stimulation by catecholamines released during neonatal hypoxia play a role in the metabolic adjustment of brain and heart tissue to damage and may aid in subsequent recovery.